Blood processing apparatus and method for detoxifying bacterial lipopolysaccharide

ABSTRACT

A detoxification method includes the steps of inducing flow of patient blood through an extracorporeal device inlet and outlet in fluid connection to the circulatory system of a patient. Biological agents including lipopolysaccharide (LPS) contained within patient blood can be detoxified by passing patient blood over a biochemical reactor surface having attached or immobilized Saccharomyces boulardii alkaline phosphatase enzyme, with the biochemical reactor being contained within the extracorporeal device. An acyloxyacyl hydrolase enzyme may also be used on the biochemical reactor surface.

RELATED APPLICATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 17/109,044, filed on Dec. 1, 2020, which is acontinuation-in-part of U.S. patent application Ser. No. 16/862,378,filed on Apr. 29, 2020, both of which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

This invention relates to detoxifying Gram negative bacteriallipopolysaccharide (LPS) and other inflammatory compounds in a person'sblood. More particularly, an external biochemical reactor containingimmobilized alkaline phosphatase enzymes for treating blood isdescribed.

BACKGROUND OF THE INVENTION

There are hundreds of different species of bacteria in thegastrointestinal (GI) tract including both beneficial bacteria (i.e.,the commensal microbiome) and potentially pathogenic bacteria. Gramnegative bacteria are one major group of potentially pathogenic bacteriain the GI tract. Gram negative bacteria have a cell wall surrounded byan outer membrane composed of lipopolysaccharides (LPS). Harmfulendotoxins can include lipoglycans and LPS, which are large moleculesconsisting of a lipid and a polysaccharide composed of O-antigen, anouter and inner protein core joined by a covalent bond, and a lipid Amoiety joined to the inner core by phosphate groups. The endotoxinsproduced by different Gram negative bacteria differ in theirantigenicity due to differences in the O-antigen, but they all have thesame biological effects which are mainly due to lipid A. Lipid Acontains two phosphate groups that are believed to be essential for itstoxicity

LPS in the bloodstream may be neutralized to some extent by many bloodcomponents including plasma lipids and proteins and LPS-binding protein(LPB). LPS binding to LPB elicits immune responses by presenting the LPSto cell surface pattern recognition receptors called CD14 and Toll-likereceptors (e.g., TLR4) on macrophages, monocytes and endothelial cells.Interaction of LPS with these cellular receptors on monocytes andmacrophages results in 1) production and release of cytokines includingtumor necrosis factor alpha (TNFα), interleukins (e.g., IL-1, IL-6,IL-8) and platelet activating factor, resulting in activation of thearachidonic acid cascade to produce prostaglandins and leukotrienes,which are potent mediators of inflammation; 2) activation of thecomplement cascade C3 and C5a resulting in release of histamine whichcauses vasodilation, inflammation, and neutrophil chemotaxis; and 3)activation of the blood coagulation cascade that leads to acutedisseminated intravascular coagulation, internal bleeding, hemorrhageand sepsis.

Sepsis is a life-threatening condition that develops when the body'sresponse to infection causes injury to its own tissues and organs.Immediate, intensive treatment is crucial for surviving the conditionand preventing septic shock because the risk of death from sepsis andseptic shock increases with every passing hour. Toxic shock occurs whenthe body has an overwhelming response to infection—sometimes referred toas a “cytokine storm”—that causes the blood pressure to drop todangerously low levels and triggers damaging changes to the organscausing them to become dysfunctional and stop working. Current treatmentstrategies include fluid replacement, antibiotics to control theinfection, vasopressors to maintain adequate blood pressure,corticosteroids and anti-inflammatory drugs to lessen inflammation, andinsulin to stabilize blood sugar levels. In some cases, a person mightrequire surgery to remove abscesses and necrotic tissues that are asource of the microbial infection and toxins.

This disclosure describes a system, apparatus and method that canaccomplish therapeutic removal of selected toxins within a biologicalsystem, including but not limited to those produced by Gram negativebacterial lipopolysaccharide (LPS) and other inflammatory compoundstoxic to humans or animals.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, a detoxification method includes the steps ofinducing flow of patient blood through an extracorporeal device inletand outlet in fluid connection to a circulatory system of a patient.Biological agents contained within patient blood can be detoxified bypassing patient blood over a biochemical reactor surface having attachedor immobilized alkaline phosphatase enzyme, with the biochemical reactorbeing contained within the extracorporeal device.

In one embodiment, the alkaline phosphatase further comprisesSaccharomyces boulardii alkaline phosphatase.

In one embodiment, the alkaline phosphatase detoxifies Gram negativebacterial lipopolysaccharide (LPS).

In one embodiment, the alkaline phosphatase detoxifies at least one ofGram negative or Gram positive bacterial extracellular lipoteichoicacid, ATP, DNA, RNA or flagellin, yeast and fungal extracellular ATP,DNA and RNA, viral extracellular DNA and RNA, and host extracellularATP, DNA, or RNA.

In one embodiment, the alkaline phosphatase detoxifies biological agentscontained within patient blood by dephosphorylation.

In one embodiment, detoxifying biological agents using S. boulardiialkaline phosphatase is used to therapeutically treat at least one ofsepsis, septic shock, inflammation, bacteremia, yeast infections, fungalinfections, viral infections, systemic inflammatory response syndrome(SIRS), Gram negative bacterial lipopolysaccharide (LPS)-exacerbatedconditions, IBD, IBS, Crohn's disease, ulcerative colitis,enterocolitis, NEC, meningitis, meningococcemia, trauma or hemorrhagicshock, burns, liver disease, pancreatitis, periodontal disease,pneumonia, cystic fibrosis, asthma, A1AT deficiency, COPD, pulmonaryfibrosis, tuberculosis, coronary heart disease, congestive heartfailure, renal disease, hemolytic uremic syndrome, kidney disease,autoimmune diseases including rheumatoid arthritis, systemic lupuserythematosus, cancer, Alzheimer's disease, diabetes, infections/abscessrelated diseases, and protein aggregation disorders includingneurodegenerative diseases, Parkinson's disease, amyloidosis, andpatients undergoing surgery, cardiovascular surgery, and transplants.

In one embodiment, alkaline phosphatase enzyme is immobilized by beingcovalently attached to the biochemical reactor surface.

In one embodiment, the biochemical reactor surface further comprises atleast one of capillary tubing and microbeads.

In one embodiment, patient blood can be pumped through an extracorporealdevice inlet and outlet in fluid connection to the circulatory system ofa patient.

In one embodiment, the biochemical reactor surface is provided with acontinuous blood flow from the patient that continues until thebiological agent(s) being detoxified have been reduced to predeterminedlevels.

In one embodiment, a blood detoxification system includes anextracorporeal device having an inlet and outlet able to be placed in influid connection to the circulatory system of a patient. A biochemicalreactor surface having attached alkaline phosphatase enzyme can act todetoxify biological agents contained within patient blood. Thebiochemical reactor can be contained within the extracorporeal device.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments will be readily understood by the following detaileddescription in conjunction with the accompanying drawings.

FIG. 1 illustrates a system including extracorporeal devices that can beattached to receive and detoxify blood or other fluids from a patient;

FIG. 2 illustrates one embodiment of a method for detoxifying patientblood;

FIG. 3 illustrates one embodiment of an extracorporeal devices that canbe attached to receive and detoxify blood that includes coiled tubingsupporting immobilized enzymes;

FIG. 4 illustrates one embodiment of an extracorporeal device that canbe attached to receive and detoxify blood that includes a flow chambersupporting beads with immobilized enzymes; and

FIG. 5 illustrates one embodiment of an extracorporeal device that canbe attached to receive and detoxify blood that is further connected toother diagnostic or therapeutic equipment.

DETAILED DESCRIPTION OF THE INVENTION

This disclosure may employ other terms and phrases not expressly definedherein. Such other terms and phrases shall have the meanings that theywould possess within the context of this disclosure to those of ordinaryskill in the art. In some instances, a term or phrase may be defined inthe singular or plural. In such instances, it is understood that anyterm in the singular may include its plural counterpart and vice versa,unless expressly indicated to the contrary.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,reference to “a substituent” encompasses a single substituent as well astwo or more substituents, and the like.

As used herein, the term “about”, when used in reference to numericalranges, cutoffs, or specific values, is used to indicate that therecited values may vary by up to as much as 10% from the listed value.As many of the numerical values used herein are experimentallydetermined, it should be understood by those skilled in the art thatsuch determinations can, and often times, will vary among differentexperiments. The values used herein should not be considered undulylimiting by virtue of this inherent variation. The term “about” is usedto encompass variations of this sort up to, or equaling, 10%.

The term “attach,” “attached” or “attachment” as used herein, refers toconnecting or uniting by a chemical bond, link, or force in order tokeep two or more chemical compounds, polymers, proteins,polysaccharides, lipids, nucleic acids, or other biological ormanufactured compositions together.

As used herein, “for example,” “for instance,” “such as,” or “including”are meant to introduce examples that further clarify a more generalsubject matter. Unless otherwise expressly indicated, such examples areprovided only as an aid for understanding embodiments illustrated in thepresent disclosure and are not meant to be limiting in any fashion. Nordo these phrases indicate any kind of preference for the disclosedembodiment.

Disclosed herein is an extracorporeal device, system or methodsinvolving circulating, perfusing, or otherwise passing blood or otherpatient fluids through a system and device external to the body. One ormore internal surfaces of the external or extracorporeal system includeimmobilized enzymatic agents to interact with one or more patient fluidborne biologic agents. The extracorporeal device, system or methodsprovide a platform that can be applied to numerous conditions anddiseases involving circulating cells, compounds, or other biologicagents, such as those associated with bacterial, yeast, fungal, or viralinfection, cell death, sepsis and many others.

FIG. 1 illustrates a system 100 that can be attached to receive blood orother fluids from a human or animal patient. The system 100 includes anextracorporeal device 110 having an inlet 112 and outlet 114. Using afluid pump 116, blood or other fluid is introduced and passed through areplaceable biochemical reactor 130. In some embodiments, thebiochemical reactor 130 can form the entirety of the extracorporealdevice 110. Surface attached and immobilized enzymatic agents in thebiochemical reactor can remove toxins or other undesired contaminantsand return the processed blood to the patient using outlet 114. Acontrol and monitoring system 140 can be used to set fluid flow rates,maintain and monitor fluid temperature, and support sensors that candetermine detoxification efficacy.

FIG. 2 illustrates one embodiment of a method 200 for detoxifying humanor animal patient blood. In a step 202, blood is drawn from a human oranimal patient using a needle and suitable arterial or venous tap orpuncture and transferred into an extracorporeal device. The patientblood is detoxified in step 204, and the process intermittently orcontinuously repeated until a significant volume of patient blood hasbeen processed and returned to the patient (step 206). Afterdetoxification, the patient can be disconnected, and the extracorporealsystem cleaned. Cleaning can include sanitization or replacement of thebiochemical reactor and readying the system for use by another patient.

In one embodiment of the device, system, or method of FIGS. 1 and 2,withdrawal of fluids from a human or animal patient can include blooddrawn by venipuncture or arterial taps. Other bodily fluids such ascerebrospinal fluid, lymph fluid, urine, stomach and GI tract fluids canalso be processed using the described systems and methods.

In one embodiment of the device, system, or method of FIGS. 1 and 2, thepump can include continuous, intermittent, or variable speed pumps.These can include but are not limited to peristaltic pump systems.

In one embodiment of the device, system, or method of FIGS. 1 and 2, theinlet and outlet can include luer locks or locking cannula systems.

In one embodiment of the device, system, or method of FIGS. 1 and 2,biochemical reactor can include fluid flow structures such as tubing,capillary tubes, hollow fibers, porous structures, and chamberscontaining unattached polymer or magnetic beads. Flow structures can beformed in whole or in part from glass, metal, ceramic, or polymericmaterials. Fluid flow structures can be continuous, split into multipleseparate flow channels using a manifold, or contain circulating closedchamber structures.

In one embodiment of the device, system, or method of FIGS. 1 and 2,immobilized enzymatic biologic agents can include alkaline phosphatase(AP), an enzyme with broad specificities that can catalyzedephosphorylation of DNA, RNA, ribo- and deoxyribonucleosidetriphosphates in humans. For example, alkaline phosphatase canenzymatically react with adenosine triphosphate to yield adenosinediphosphate and a free phosphate. Under biological conditions, alkalinephosphatase can remove phosphate from phosphate containing biologicagents such as nucleotides and proteins, inactivating or detoxifying thebiologic agents.

Various types or isozymes of alkaline phosphatase can be used, includingbut not limited to human or animal derived intestinal AP (IAP),tissue-nonspecific AP (ALPL), placental AP (ALPP), germ cell AP (GCAP),or yeast derived alkaline phosphatase. Because of wide availability,ease of culture, and long duration of enzymatic activity, alkalinephosphatase produced by strains of the yeast Saccharomyces cerevisiaevar. boulardii (S. boulardii) can be used in one embodiment.Advantageously, S. boulardii alkaline phosphatase has excellentstability and active lifetime, a pH activity profile suited fordephosphorylating toxins at normal pH blood, is less expensive toprepare than human or animal sources of alkaline phosphatase, andman-made alkaline phosphatase such as that made using recombinant DNAtechnology

In one embodiment of the device, system, or method of FIGS. 1 and 2,attachment of enzymatic biologic agents such as alkaline phosphatase(AP) can include linkage using conventional affinity tag binding,attachment or adsorption on glass, beads, alginate structures or othermatrix, entrapment in insoluble beads or microspheres, enzymatic crosslinkage to create an enzymatically reactive surface, or covalentbonding. Covalent bonding can be random or site specific. Amino, thiol,carboxyl, or cyanogen bromide activation can be used. In someembodiments, discrete linking agents that are attached between thealkaline phosphatase and a surface can be used. In some embodiments,surfaces can be chemically modified to allow enzyme attachment, orfunctional groups exposed on the surface can be activated. Covalent orionic coupling a linking agent or enzyme to the surface can includelinking of one or more functional groups on the surface or the enzyme.

In one embodiment of the device, system, or method of FIGS. 1 and 2,biologic agents or compounds that interact with the immobilizedenzymatic agents can include blood or fluid conveyed LPS, as well asadenosine triphosphate (ATP), DNA, RNA and flagellin. More specifically,in other embodiments, at least one of Gram negative or Gram positivebacterial extracellular lipoteichoic acid, ATP, DNA, RNA or flagellin,yeast and fungal extracellular ATP, DNA and RNA, viral extracellular DNAand RNA, and host extracellular ATP, DNA, or RNA can be detoxified afterinteraction with immobilized alkaline phosphatase.

In one embodiment of the device, system, or method of FIGS. 1 and 2,treatable diseases, conditions, or symptoms of humans or animals caninclude but are not limited to:

1) Stand-alone treatment or in conjunction with other treatmentstrategies for bacteremia, sepsis and septic shock including oral dosingand IV injection of IAP or other AP isozymes;

2) Treatment to lessen morbidity due to LPS translocating from thegastrointestinal tract into the bloodstream when the intestinalpermeability barrier becomes compromised due to dysbiosis caused by Gramnegative bacterial infection and/or other acute or chronic metabolicdisorders such as IBD, inflammatory bowel syndrome (IBS), Crohn'sdisease, cancer, liver disease, autoimmune diseases, diabetes, or aging;3) Treatment to detoxify LPS exogenous ATP, DNA and flagellin in thebloodstream due to translocation across the intestinal barrier or due tomicrobial infections, bacteremia, abscesses or tissue necrosis at anysite in the body;4) Treatment to detoxify LPS exogenous ATP, DNA and flagellin in thebloodstream due to translocation following antibiotic treatment thatalters the gut microbiome and disrupts homeostasis to allow increasedcontact of Gram negative bacteria in the intestinal lumen to gain accessto Toll-like receptors on enterocytes, resulting in inflammation anddecreased intestinal barrier function. In some situations, S. boulardiiprobiotics and/or oral IAP can additionally be used to accelerate returnto normal homeostasis in the gastrointestinal tract;5) Treatment to reduce the inflammatory response associated withnecrotizing enterocolitis (NEC) and mitigate the septic response andend-organ injury;6) Treatment of sepsis, septic shock, inflammation, bacteremia, yeastinfections, fungal infections, viral infections, systemic inflammatoryresponse syndrome (SIRS), Gram negative bacterial lipopolysaccharide(LPS)-exacerbated conditions, IBD, IBS, Crohn's disease, ulcerativecolitis, enterocolitis, NEC, meningitis, meningococcemia, trauma orhemorrhagic shock, burns, liver disease, pancreatitis, periodontaldisease, pneumonia, cystic fibrosis, asthma, A1AT deficiency, COPD,pulmonary fibrosis, tuberculosis, coronary heart disease, congestiveheart failure, renal disease, hemolytic uremic syndrome, kidney disease,autoimmune diseases including rheumatoid arthritis, systemic lupuserythematosus, mast cell activation disorders, cancer, Alzheimer'sdisease, diabetes, infections/abscess related diseases, and proteinaggregation disorders including neurodegenerative diseases, Parkinson'sdisease, amyloidosis, and patients undergoing surgery, cardiovascularsurgery, and transplants;7) Treatment of conditions associated with extracellular ATP, includinghypoxia and ischemia that result in active release from cells andpassive leakage from damaged/dying cells, and downregulation ofectonucleotidases. Examples include but are not limited to SIRS, A1AT,COPD, IBD, IBS, diverticulosis, and diverticulitis;8) Treatment of proinflammatory conditions derived from increased ATP,DNA and flagellin in patients with IBD, ulcerative colitis, or otherdisorder in which there is a decreased expression of IAP that results inexcessive levels of proinflammatory compounds in the bloodstream;9) Treatment to reduce levels of LPS, DNA, extracellular ATP andflagellin in the bloodstream that may be elevated following a course ofsystemic antibiotic treatment that altered the GI microbiome to favornon-commensal Gram negative bacteria including Escherichia coli,Citrobacter freundii, Enterobacter aerogenes, and other bacteria,resulting in increased translocation of LPS, DNA, external ATP andflagellin into the bloodstream. Reduction of these toxins in thebloodstream can reduce immunological response and enable the body tore-establish the normal commensal microflora and homeostasis in the Gitract; and10) Treatment to reduce toxicity of LPS and microbial or host chemicalcompounds that may be proinflammatory including lipoteichoic acid, ATP,DNA and flagellin to reduce patient morbidity and result in fewerfinger, hand, toe, foot, arm and leg amputations and surgery caused bysepsis and inflammation.

FIG. 3 illustrates one embodiment of an extracorporeal device that canbe attached to receive and detoxify blood that includes coiled tubingsupporting immobilized enzymes. As seen in FIG. 3, a system 300 can beattached to receive blood from vein connections to a patient's arm. Thesystem 300 includes an extracorporeal device 310 having a luer lockinlet 312 and luer lock outlet 314. Using a fluid pump 316, blood orother fluid is introduced and passed through a replaceable biochemicalreactor 330 that includes coiled or otherwise compactified tubing.Surface attached and immobilized enzymatic agents in the biochemicalreactor can remove toxins or other undesired contaminants and return theprocessed blood to the patient using outlet 314. A control andmonitoring system 340 can be used to set fluid flow rates, maintain andmonitor fluid temperature, and support sensors that can determinedetoxification efficacy. In this embodiment, a heater element 332 can beconnected to the control and monitor device to maintain bloodtemperature at about 37 degrees Celsius for human patients, or normalblood temperature for non-human patients.

FIG. 4 illustrates one embodiment of an extracorporeal device that canbe attached to receive and detoxify blood that includes a flow chambersupporting beads with immobilized enzymes. As seen in FIG. 4, a system400 can be attached to receive blood or other fluids from arteries orvein connections to a patient's arm. The system 400 includes anextracorporeal device 410 having a luer lock inlet 412 and luer lockoutlet 414. Using a fluid pump 416, blood or other fluid is introducedand passed through a replaceable biochemical reactor 430 that includes afluid chamber partially filled with beads capable of surface attachmentwith immobilized enzymatic agents. A magnetic stirring system 434 can beused to rotate magnetic cylinders 436 within the replaceable biochemicalreactor 430. Surface attached and immobilized enzymatic agents in thebiochemical reactor can remove toxins or other undesired contaminantsand return the processed blood to the patient using outlet 414. Acontrol and monitoring system 440 can be used to set fluid flow rates,maintain and monitor fluid temperature, and support sensors that candetermine detoxification efficacy. In this embodiment, a heater element432 can be connected to the control and monitor device to maintain bloodtemperature at a desired set point.

FIG. 5 illustrates one embodiment of an extracorporeal device that canbe attached to receive and detoxify blood that is further connected toother diagnostic or therapeutic equipment. As seen in FIG. 5, a system500 can be attached to receive blood or other fluids from arteries orvein connections to a patient's arm. The system 500 includes anextracorporeal device 510 having a luer lock inlet 512 and luer lockoutlet 514. Using a fluid pump 516, blood or other fluid is introducedand passed through a replaceable biochemical reactor 530 that includesimmobilized enzymatic agents such as alkaline phosphatase. After passingthrough the biochemical reactor 530, blood can be further processed byseparate (as shown) or built-in diagnostic or therapeutic systems 540.Diagnostic systems can include inline assays for free phosphate,real-time flow cytometry, or other blood health diagnostics. Therapeuticsystems can include additional hemoperfusion, hemofiltration,oxygenation, or other blood processing methods. In some embodiments,diagnostic or therapeutic systems 540 can include in-line sampling portsthat permit periodic sample taking.

Various modifications to the foregoing described embodiments can bemade. For example, multiple glass or plastic capillary tubing reactionchambers can be connected in series to act as biochemical reactors. Inone embodiment, a first capillary tubing can contain immobilized SBAP toirreversibly dephosphorylate LPS, ATP and other proinflammatorycompounds, and a second capillary tubing provided that containsimmobilized Apyrase/CD73 to complete dephosphorylation of AMP toadenosine and inorganic phosphate if this reaction has not beencompleted in the first reaction chamber In other embodiments, areplaceable biochemical reactor can be used. The replaceable biochemicalreactor can include immobilized SBAP or other alkaline phosphatasecovalently attached to magnetic microbeads that are held by magneticattraction to the inner surface of capillary tube. In still otherembodiments magnetic microbeads with covalently attached enzymaticagents including SBAP and apyrase can be used in conjunction with amagnetic stirring system that is used to rotate magnetic cylinderswithin the biochemical reactor. Stirring cylinders can include surfaceattached and immobilized enzymatic agents to detoxify toxins or otherundesired contaminants in the blood of the patient as the blood ispumped continuously through the biochemical reactor.

As will be appreciated, the described systems and methods of FIGS. 3, 4,and 5 that are applicable to venous blood taken and returned to apatient arm can be adapted to process fluids from other body sites. Suchbodily fluids may be routed to the extracorporeal device fordetoxification and then returned to the body. In other embodiments,instead of removal of toxins, toxins can instead or additionally bereversibly or irreversibly detoxified or dephosphorylated. This canapply to toxins including LPS, Gram negative or Gram positive bacterialextracellular lipoteichoic acid, nucleotides including ATP, ADP, DNA,RNA and flagellin, yeast and fungal extracellular ATP, ADP, DNA, andRNA, viral DNA and RNA, and host extracellular nucleotides includingATP, ADP, DNA, and RNA.

Although AP dephosphorylation of LPS has been considered to protect thehost from systemic stimulation due to translocated intestinal LPS,recent findings by Komazin et al. (Komazin, G. et al. Substratestructure-activity relationship reveals a limited lipopolysaccharidechemotype range for intestinal alkaline phosphatase. J. Biol. Chem.294(50):19405-19423 (2019)) revealed that certain primary (glucosaminelinked) side chains of LPS must be removed from lipid A beforeintestinal LPS can cleave either of the lipid A phosphate groups.Because of recent findings, Munford et al. (Munford, R. S., J. P. Weissand M. Lu. Biochemical transformation of bacterial lipopolysaccharidesby acyloxyacyl hydrolase reduces host injury and promotes recovery. J.Biol. Chem. 295(51):17842-17851 (2020)) observed that acyloxyacylhydrolase (AOAH) conversion of LPS to deacylated LPS (dLPS) has becomethe most likely mechanism for reducing the stimulatory potency of LPS inthe intestine. This conversion transforms LPS from a stimulus ofinflammation to dLPS which reduces tissue injury and death from Gramnegative bacterial infection.

In addition to alkaline phosphatase enzymes such as previouslydiscussed, other phosphatase enzymes including any of human knownalkaline phosphatase (AP) isozymes including intestinal AP (IAP),tissue-nonspecific AP (TNAP), placental AP (PLAP), and germ cell AP(GCAP), any of other human phosphatase, AP, or nucleotidases includingapyrase/CD39, CD73 (ecto-5′-nucleotidase), any of synthetic or man-madeAP, such as AP made from recombinant DNA including E. coli, any of humanor animal apyrase (cluster of differentiation 39=CD39) or AP and humannucleotidases human cluster of differentiation 73(CD73)/ecto-5′-nucleotidase, bovine alkaline phosphatase (blAP), calfalkaline phosphatase (clAP), potato apyrase, and AP from shrimp andrecombinant DNA technology (E. coli alkaline phosphatase) can be used.Addition of AOAH to the phosphatase enzymes would help insuredestruction of all forms of circulating LPS in the bloodstream.

In some embodiments, an extracorporeal device with attached phosphatasebiochemical reactor can be used as a stand-alone device or inconjunction with other treatment modalities. This can include but is notlimited to devices or treatments including fluid replacement,corticosteroids, oral and IV administration of APs for treatment ofsepsis, treatment of cytokine storm caused by serious acute respiratoryvirus SARS Covid-19 infection, injured/diseased organs and tissues,chronic inflammatory diseases such as diabetes and COPD, and followingsurgery, to reduce inflammation and speed recovery. As will beunderstood, treatment for Gram negative bacterial lipopolysaccharide(LPS) in patient blood, infection resulting in LPS or lipoteichoic acidin patient blood, abscesses resulting in LPS or lipoteichoic acid inpatient blood, or other toxins in patient blood such as discussed hereinare also contemplated.

Example 1

In one example embodiment, a sterile hypodermic needle set can be usedfor accessing a patient's vein, (e.g., Blood Collection Set, Vaculet21Gx3/4″ Winged, w/Multi-Sample Adapter, 12″ Tubing, or similar veinaccessing device with a larger bore needle, if needed). A 36″ length ofsterile plastic tubing can be used to pass through a peristaltic pumpand connect the Vaculet with the biochemical reactor with a luer lock.The peristaltic pump or similar pumping device can be used for pumpingblood from the patients arm to the biochemical reactor.

An external continuous-flow biochemical reactor is prepared byimmobilizing S. boulardii alkaline phosphatase (SBAP) on the innersurface of polystyrene, polymethacrylate, or other plastic capillarytubing as described by Habja and Guttman “Continuous-flow biochemicalreactors: Biocatalysis, bioconversion, and bioanalytical applicationsusing immobilized microfluidic enzyme reactors”. J. Flow. Chem.6(1):8-12, 2015, or Mohamad, et al. “An overview of technologies forimmobilization of enzymes and surface analysis techniques forimmobilized enzymes. Biotech. Biotechnol. Equip. 29(2):205-220, 2015.Enough capillary tubing (i.e., preferably up to 36 inches long) is usedto allow immobilization of 50-1,500 IU AP, and preferably 200-500 IUSBAP in the tubing, which becomes the biochemical reactor. The plastictubing with immobilized SBAP on the inner surface is sterilized and maybe stored in the refrigerator at 4° C. for several months prior to use.When ready for use, the plastic tubing is placed into a 12″×12″×12″chamber that has a lid that opens for placement of the tubing inside, aside opening with a luer lock for connection to the plastic tubing fromthe patient's arm (by way of the peristaltic pump), and a second sideopening with a luer lock for connecting to the line that returns bloodto the patient. Alternatively, the plastic tubing can be used withoutinsertion into a chamber, with the pump and tubing together forming aportion of the extracorporeal device by themselves. The continuous-flowbiochemical reactor can dephosphorylate approximately 50% of the LPS,external ATP, DNA and flagellin in blood per passage through it as theblood is pumped slowly (e.g., flow rate of 0.5-50 mL/min, and typicallyup to 10 mL/min) through the capillary tubing before returning it to thepatient. Passage of the patient's blood through the biochemical reactordephosphorylates and thereby detoxifies LPS, Gram negative or Grampositive bacterial extracellular ATP, DNA, RNA and flagellin, and hostextracellular ATP, DNA, and RNA in the patient's blood before it isreturned to the patient

Example 2

A sterile hypodermic needle set can be used for accessing a patient'svein and a peristaltic pump can be used for pumping blood from thepatient's arm to a biochemical reactor. An external continuous-flowbiochemical reactor is prepared by use of a sterile 250 mL closedcontainer with a magnetic stirring bar that contains 10-150 g, andpreferably 50 g of microbeads with immobilized SBAP, prepared bycovalent bonding to have 50-1,500 IU SBAP, and preferably 200-500 IUSBAP prepared aseptically in the biochemical reactor. The externalcontinuous-flow biochemical reactor has inlet and outlet connections forconnecting with blood being pumped to and from the continuous-flowbiochemical reactor. The container with SBAP covalently immobilized onthe plastic beads is sterilized and may be stored in the refrigerator at4° C. for several months prior to use. When ready for use, the containeris placed onto a magnetic stirrer and stirring is started when bloodbegins to fill the container. A length of sterile plastic tubing is usedto connect the biochemical reactor to the patient's arm vein forreturning treated blood to the patient's arm or leg vein.

The peristaltic pump may be turned on after checking to ensure that allconnections are tight so that they will not leak or allow the blood tobecome contaminated, and the pump is run continuously. Passage of thepatient's blood through the biochemical reactor dephosphorylates andthereby detoxifies LPS, Gram negative or Gram positive bacterialextracellular lipoteichoic acid, ATP, DNA, RNA and flagellin, and hostextracellular ATP, DNA, and RNA in the patient's blood before it isreturned to the patient.

The biochemical reactor chamber is maintained at approximately bodytemperature (37° C.) by use of a thermostatically-controlled heatingdevice. The patient's blood is pumped continuously through thebiochemical reactor until analytical testing shows that levels of theLPS and inflammatory chemicals including, but not limited to ATP, DNAand flagellin, and/or selected markers (e.g., cytokines including TNFα,IL-6, or IL-8) have been reduced to undetectable or baseline levels andthe patient's signs have returned to normal.

While specific embodiments have been illustrated and described above, itis to be understood that the disclosure provided is not limited to theprecise configuration, steps, and components disclosed. Variousmodifications, changes, and variations apparent to those of skill in theart may be made in the arrangement, operation, and details of themethods and systems disclosed, with the aid of the present disclosure.

Without further elaboration, it is believed that one skilled in the artcan use the preceding description to utilize the present disclosure toits fullest extent. The examples and embodiments disclosed herein are tobe construed as merely illustrative and exemplary and not a limitationof the scope of the present disclosure in any way. It will be apparentto those having skill in the art that changes may be made to the detailsof the above-described embodiments without departing from the underlyingprinciples of the disclosure herein.

The invention claimed is:
 1. A detoxification method, comprising:inducing flow of blood of a patient through an extracorporeal deviceinlet and outlet; and detoxifying at least one biological agentcontained within the blood by passing the blood over at least onebiochemical reactor surface having attached phosphatase enzyme andacyloxyacyl hydrolase enzyme, with the at least one biochemical reactorsurface being contained within the extracorporeal device and wherein theat least one biological agent is selected from the group consisting of:Gram negative bacterial lipopolysaccharide (LPS); Gram negativebacterial flagellin; Gram positive bacterial extracellular lipoteichoicacid; Gram positive bacterial flagellin; one or more bacterialextracellular nucleotides selected from adenosine triphosphate (ATP),adenosine diphosphate (ADP), deoxyribonucleic acid (DNA), andribonucleic acid (RNA), one or more yeast extracellular nucleotidesselected from ATP, ADP, DNA and RNA, one or more fungal extracellularnucleotides selected from ATP, ADP, DNA and RNA, one or more viralextracellular nucleotides selected from DNA and RNA, and one or morehost extracellular nucleotides selected from ATP, ADP, DNA, and RNA, andcombinations thereof.
 2. The detoxification method of claim 1, furthercomprising connecting the extracorporeal device inlet and outlet to thepatient.
 3. The detoxification method of claim 1, wherein inducing theflow of the blood of the patient through the extracorporeal device inletand outlet is performed continuously.
 4. The detoxification method ofclaim 1, wherein the inducing the flow of blood of the patient throughthe extracorporeal device inlet and outlet comprises removing batches ofblood from the patient and, for each batch of the batches of blood,passing the batch through the extracorporeal device inlet and outlet andreturning the batch to the patient.
 5. The detoxification method ofclaim 1, wherein the phosphatase enzyme detoxifies the at least onebiological agents contained within the blood by dephosphorylation. 6.The detoxification method of claim 1, wherein the acyloxyacyl hydrolaseenzyme detoxifies the at least one biological agent contained within theblood by deacylation.
 7. The detoxification method of claim 1, whereindetoxifying the at least one biological agent contained within the bloodof the patient therapeutically treats at least one of sepsis, septicshock, inflammation, bacteremia, a yeast infection, a fungal infection,a viral infections, systemic inflammatory response syndrome (SIRS), Gramnegative bacterial lipopolysaccharide (LPS) in the blood, IBD, IBS,Crohn's disease, ulcerative colitis, enterocolitis, NEC, meningitis,meningococcemia, trauma, hemorrhagic shock, a burns, liver disease,pancreatitis, periodontal disease, pneumonia, cystic fibrosis, asthma,A1AT deficiency, COPD, pulmonary fibrosis, tuberculosis, coronary heartdisease, congestive heart failure, renal disease, hemolytic uremicsyndrome, kidney disease, an autoimmune diseases, rheumatoid arthritis,systemic lupus erythematosus, a mast cell activation disorders, cancer,Alzheimer's disease, diabetes, infection resulting in LPS orlipoteichoic acid in the blood, an abscess resulting in LPS orlipoteichoic acid in the blood, a protein aggregation disorder, aneurodegenerative disease, Parkinson's disease, amyloidosis, and acondition following surgery.
 8. The detoxification method of claim 1,wherein the phosphatase enzyme and the acyloxyacyl hydrolase enzyme arecovalently attached to the at least one biochemical reactor surface. 9.The detoxification method of claim 1, wherein the at least onebiochemical reactor surface comprises at least one of one or moresurfaces of capillary tubing and surfaces of microbeads.
 10. Thedetoxification method of claim 1, wherein the at least one biochemicalreactor surface comprises surfaces of magnetic microbeads.
 11. Thedetoxification method of claim 1, wherein the at least one biochemicalreactor surface is provided with continuous flow of the blood thatcontinues until the at least one biological agents being detoxified haseach been reduced to predetermined levels in the blood.